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Mitochondrial biogenesis as revealed by mitochondrial transcript profiles during germination and early seedling growth in wheat. Sakina M. Khanam1†, Nayden ...
Genes Genet. Syst. (2007) 82, p. 409–420

Mitochondrial biogenesis as revealed by mitochondrial transcript profiles during germination and early seedling growth in wheat Sakina M. Khanam1†, Nayden G. Naydenov1†, Koh-ichi Kadowaki2 and Chiharu Nakamura1*† 1

Laboratory of Plant Genetics, Department of Agroenvironmental Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan 2 Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan (Received 19 June 2007, accepted 6 September 2007)

Germination of imbibed embryos is the initial stage of plant development that is accompanied by the burst of mitochondrial respiration. To understand the process of mitochondrial biogenesis during this critical stage in wheat development, we monitored changes in mitochondrial transcript profiles during the first 3 days by adopting a newly devised macroarray system. The whole experiment was conducted in the dark to avoid influences of photosynthesis. Dry quiescent embryos started respiration rapidly after imbibition and the rate of oxygen uptake increased to peak at the first day followed by a continuous decrease until the third day under this condition. Both the cytochrome and alternative electron transport pathways appeared to contribute to this initial burst. Shoot and root growth was also remarkable during this period. Mitochondrial transcriptome was studied by macroarray analysis using 28 mitochondrial protein-coding genes, 4 nuclear encoded mitochondria-targeted genes and 2 nuclear genes as control. All transcripts were present in dry embryos at different initial levels, and a large variability was observed in their abundance among individual genes throughout the tested period. Gene expression was categorized into four clusters according to the profiles of individual transcript accumulation. A majority of the genes encoding subunits of the respiratory complexes belonged to two major clusters, the time course of transcript accumulation of one cluster agreeing with that of respiratory development and the other remaining at high constant levels. The macroarray system devised in this study should be useful in monitoring mitochondrial biogenesis under various growth conditions and at different developmental stages in cereals. Key words: germination, macroarray, mitochondrial biogenesis, transcript profiling, wheat (Triticum aestivum L.) INTRODUCTION Mitochondria play essential roles in energy production through oxidative phosphorylation, and participate in various metabolic pathways and also cell death in both plants and animals (Reichert and Neupert, 2004). Glycolysis, tricarboxylic acid cycle and mitochondrial electron transport chains are all located in this intracellular organella (Fernie et al., 2004). Seed germination is one of the most critical stages of plant development, in which mitochondria play a prominent role as ATP and subEdited by Toru Terachi * Corresponding author. E-mail: [email protected] † these authors contributed equally to this work

strates for various biosynthetic pathways are supplied almost exclusively by mitochondria. In the process of seed germination, imbibed embryos undergo rapid transition from dry quiescent state into hydrated and rapidly respiring state (Bewley and Black, 1994). This developmental stage therefore provides a good opportunity to study mitochondrial biogenesis, i.e. structural and functional development of mitochondria (Leaver and Lonsdale, 1989). Mitochondrial biogenesis during germination and early seedling growth has been studied in peanut (Morohashi et al., 1981), cucumber (Hill et al., 1992), maize (Ehrenshaft and Brambl, 1990; Li et al., 1996; Logan et al., 2001) and rice (Howell et al., 2006, 2007). It has been reported that poorly differentiated but fully

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functional mitochondria are present in dry sunflower embryos (Attucci et al., 1991). Such protomitochondria, upon imbibition, enlarge and elaborate a more complex inner membrane structure in peanut (Morohashi et al., 1981) and in rice (Howell et al., 2006). Two types of mitochondria were isolated from germinating maize embryos: one type (heavy mitochondria) probably represents newly developing ones, and the other (light mitochondria) represents degraded forms that were active during embryo formation (Logan et al., 2001). Mitochondrial gene expression was studied during wheat leaf development (Topping and Leaver, 1990), sunflower anther development (Moneger et al., 1994; Smart et al., 1994), and soybean cotyledon development (Daley et al., 2003). However, a very few study describes the start of plant germination from the energy production aspects and, to our knowledge, there is a very few comprehensive study on the mitochondrial transcriptome during this critical transition stage (Li-Pook-Than et al., 2004; Howell et al., 2006). The mitochondrial genome plays important roles in plant development and productivity (Miller and Koeppe, 1971: Dewey et al., 1987; Siculella and Palmer, 1988). Plant mitochondrial genome encodes significantly more genes than do their fungal and animal counterparts (Leaver and Gray, 1982: Levings and Brown, 1989). However, while the estimated 2,000 to 3,000 proteins constitute the functional plant mitochondria (Millar et al., 2004, Moller and Gardestrom, 2007), wheat mitochondrial genome, for example, encodes only 55 genes including 35 protein coding genes (18 respiratory subunits, 11 ribosomal proteins, 4 cytochrome C biogenesis, 1 maturase related protein matR and 1 translocase protein mttB), 3 rRNA genes and 17 tRNA genes (Ogihara et al., 2005). Most of the genes encoding mitochondrial protein subunits have been transferred from the mitochondrial genome to the nuclear genome during evolution (Adams and Palmer, 2003), and the regulation of mitochondrial gene expression is under control by communication between nuclear and mitochondrial genomes. Mitochondria also communicate with chloroplasts, another intracellular organella. Therefore, analysis of organellar transcriptome can potentially identify genes that are controlled by unique or common nuclear factors (Leister, 2005: Duke et al., 2006). Micro- and macroarray technology is gaining increasing popularity because of its high capacity of simultaneous analysis of a large number of transcripts. cDNA microarray has been used to monitor mitochondrial gene expression in Arabidopsis cell culture (Giege et al., 2005). cDNA macroarray system also has emerged to be a tool in gene expression profiling of nuclear (Hirai et al., 2003; Ji et al., 2003), mitochondrial (Okada and Brennicke, 2006) and chloroplast transcripts (Legen et al., 2002). In the present study, we devised a macroarray system and

monitored the dynamics of wheat mitochondrial transcriptome during the initial 3 days after germination to understand mitochondrial biogenesis at the level of gene expression. MATERIALS AND METHODS Plant materials and conditions of bioassay Common wheat (Triticum aestivum L.) cv. Chinese Spring (abbreviated as CS) was used throughout the study. Seeds were imbibed for 5 h under tap water and kept at 4°C overnight (~ 15 h) to promote synchronous germination. Imbibed seeds were placed on moist filter papers containing 0.1% Hyponex solution (N-P-K = 6-10-5, Hyponex, Osaka, Japan) in petri dishes and kept in the darkness at 25°C. Protrusion of coleoptiles and radicles started 9–12 h after transfer to petri dishes, and only embryos initiating germination were subjected to the experiment. Fresh weight and length of shoot and root were measured after one, two and three days. The whole experiment was repeated three times with 10 germinating seeds per experiment. Measurement of respiration Dry embryos dissected from seeds were directly subjected to respiratory measurement. Germinating embryos and seedlings until 3 days after imbibition were also used for respiratory measurement. Before the measurement, vacuum infiltration was applied with buffer solution (10 mM Mes-50 mM Hepes, pH 6.6) for 2–3 min to allow penetration of oxygen and respiratory inhibitors into the tissues. The rate of oxygen uptake was measured in the dark by placing the excised embryos or shoots cut into pieces directly in 3 ml of buffer solution in a cell with a Clark-type oxygen electrode (Yellow Springs Instrument, USA). The capacity of the cytochrome pathway was estimated as the rate of oxygen uptake inhibited by 2 mM potassium cyanide (KCN) in the presence of 2 μM n-propyl gallate (nPG), while that of the cyanide-insensitive alternative pathway by the rate inhibited by 2 μM nPG in the presence of 2 mM KCN. KCN was dissolved in distilled water and nPG in ethanol. Macroarray analysis of transcripts encoding mitochondiral proteins For constructing primers in the macroarray analysis of wheat mitochondrial transcripts, we used sequence information of mitochondrial genomes of rice (Notsu et al., 2002) and wheat (Ogihara et al., 2005). All protein-coding genes of rice mitochondrial genome are commonly present in wheat, although rps19 and rpl2 are functional in rice but not in wheat and thus these were not included in this study. We selected 21 genes encoding proteins with known function and 1 open reading frame (orf176) from the rice mitochondrial genome. Four mitochondrial protein-coding genes (atp4,

Mitochondrial biogenesis and transcript profiles Table 1.

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Primers used for the amplification of gene fragments for macroarray analysis

Gene Forward name (5’–3’) Mitochondria –encoded genes

Reverse (5’–3’)

Annealing (°C) Temperature

Product size(bp)

Position in mt. genome*

Rice genes* nad1** nad2** nad3 nad4 nad4l nad5**

aaaagcacggacgagccaca caaatccgaagaagttatca agattgagtcgccgctatcact gcacagaaagacccctattg aaaaatccaaataggctctc ctgatctatctactcggcaa

agtccccgagacattggctt gggtctctacaagtctataa ctaccatgacgaatcaattga acatagggattggcacgctt aagagaacgaaaggaaaaca gtctcgggtcttatatgctt

57 53 55 53 54 53

314 533 642 555 450 365

nad6 nad7 nad9

aacattcaagttccatttcaag agtttcgttgttcgttccgt cttagagcaagaagcggaacca

gtggactcgaaccacaattctc accactgaatccccaatcct ttgtctcctggactagactagt

55 54 55

680 483 658

cob cox1 cox2 cox3

agttgtcacgatagaaaaga tggtttagtatcaaggttct gcagagctaaaaaagatggg ctcacttctatcaatgcaatg

aaataagggggagtaaatga gggcaatagttaggagaggtgcgc tcgttgcacttaaatcaccc cacgagtgatttatcataaca

53 53 53 53

609 1680 452 1023

atp1

tatgatctagtggagtgagtga

tttcgtgatcgaatgacgaa

53

1662

atp6

acatcgatgaagctagcacga

gacttacttcgcttcgctatcta

56

546

atp9 ccmB ccmC ccmFn matR

gaaaagcgtgacgagcaaag ctagcgtgcgccagccgtcga atcactatatagtctttcttgt gatcatcctgtggttaccggatg ggctttgctccccttttttt

cactcttctttcccccacca gtactcgctgaacttacatac tcgagcttctatttcttccgt ctattcctattgatcagaagtat gttgctttgcgttggatgct

54 55 53 53 57

356 762 746 1904 628

Rice ORFs orf25***

gtctccctttctcttttgtt

tgaaaagtgaaaacctgtaa

53

531

orf176

gttcggaatccaatccagttga

cgaatcgtgaatacatgtta

55

621

Wheat genes* atp4*** agaaactctcgacgggagaa atp8 ccgaagtatcccaatggtgt

cgggaagaagtggcatttag tccaacaagtgatccgactg

56 56

252 171

ccmFc mttB rps7 rps12

gattttctgcatcggtggat tggttctcgcttctccttgt cagtatctcagcaaatgaaca ccaaaagttccagaggcatc

ccgatgaagccaacagaaat agctgtgaaaagcggaaaaa ttctcctttcctttctcttttc caaaggggggaaggacatag

55 55 55 57

253 285 470 433

196989–196738 (C) 233616–233446 (C) 339107–338937 (C) 100520–100772 315357–315642 380148–379680 (C) 341083–340652(C)

Wheat ORFs orf349 orf359

agtttatttcagtcgtattc gaggaagaaatgatgaccac

ttttctttttctcttttttg gaggttagtggcagcattat

50 51

709 693

212615–213323 102124–102816

55 55 55 55 68

650 500 400 100 1119

Wheat nuclear-encoded mitochondria-targeted genes Whlp taggcagagagcagggtgaa ccttgagatgtccattttcc TaMRPL5 gccgctccacagtagtaaag aaactgaatcacgccaatgt TaMRPL11 gccgtgccgatgacggactt acaatccgactcctagttcc MnSOD cagagggtgctgctttacaa ggtcacaagagggtcctgat Waox 1a gattgtgattcgcggaggcgttc tagctctcatttcctctgccttcc

240147–239833 (C) 404259–403727 (C) 175307–175948 198290–198844 298738–299187 115267–114902 (C), 20739–20375 (C) 60173–59494 (C) 88570–88088(C) 361745–362402, 442832–443489 305064–304456 (C) 338492–340171 212385–212836 18161–17139 (C), 112688–111666(C) 352283–353944, 433370–435031 26837–26292 (C), 121364–120819(C) 263101–263456 391698–392459 61180–60434 (C) 323917–322014 (C) 317874–317247 (C)

19052–18522 (C), 113579–113049(C) 255334–254714 (C)

Wheat nuclear-encoded genes Ubiquitin ggagcttactggccac gcatgcagatatttgtgaa 50 450 Actin ggctggttttgctggtgacgat aatgaaggaaggctggaagagga 55 600 * Primer positions for rice genes are from the database of japonica cultivar “Nipponbare” (BA 000029) and those for wheat from the database of CS (NC 007579). C indicates genes that are encoded by the complementary strand. ** These are trans-split genes. Exon 3 was used for nad1, exon 2 for nad2 and exon 1 for nad5. *** orf25 of rice and atp4 of wheat are identical gene.

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atp8, ccmFc, mttB) and 2 orfs (orf349 and orf359) from wheat were also included. orf349 is present solely in wheat and no homologous sequences can be found by blast search in other plant mitochondrial genomes. All orfs smaller than 300-bp, tRNA, rRNA and ribosomal protein genes were excluded except for wheat rps7 and rps12. In addition, 5 nuclear encoded and mitochondria-targeted wheat genes (Mn-SOD, Whlp, TaMRPL5, TaMRPL11, Waox1a) and 2 nuclear encoded genes (Actin and Ubiquitin as control) were included. A majority of the primers used were constructed based on the rice mitochondrial genome sequences (Table 1). Primer sequences for wheat rps7 and rps12 were after Tsukamoto et al. (2000), for Mn-SOD after Baek and Skinner (2003), for Whlp, TaMRPL5 and TaMRPL11 after Mizumoto et al. (2004) and for Waox1a from Takumi et al. (2002). These rice and wheat genes were amplified from DNA of “Nipponbare” and “CS”, respectively, by PCR using rTag DNA polymerase (TOYOBO, Osaka, Japan). Thirty-five cycles of PCR were performed using GeneAmp® PCR System 9700 (Perkin Elmer/Cetus). The PCR program was as follows: a denaturation step for 1 min at 94°C, an annealing step for 1 min at different temperatures (Table 1) and an extension step for 1 min at 72°C. The amplified fragments were fractionated by electrophoresis through 0.8% agarose gel and visualized by staining with ethidium bromide for size verification. Excess primers were removed by microSpin S-400 MR column (GE Healthcare) according to the manufacturer’s protocol. After estimating concentrations of PCR products, 50 ng of each was dissolved in total volume of 6 μl water and an equal volume of 20 × SSC was added. PCR products were spotted six times manually in 2 μl each time onto nylon membrane (7.5 × 8.5 cm2; HybondTM–N+). After spotting, membranes were subjected to denaturation for 30 sec (1.5 M NaCl-0.5 M NaOH) and 1 min (1.5 M NaCl, 0.5 M Tris-HCl pH 7.2, 0.1mM Na2EDTA), neutralization for 20 min (0.4N NaOH), and washing for 1 min (5 × SSC). After drying at room temperature, membranes were baked for 2 h at 80°C to immobilize DNA onto the membrane surface. RNA isolation and probe preparation Total RNA was isolated directly from dry embryos and also from germinating embryos and growing shoots according to the guanidium thiocyanate method (Cephasol RNA-I, Nacalai Tesque, Kyoto, Japan). Total RNA (2 μg) was reverse transcribed by the first strand cDNA synthesis kit, Rever TRA ACE (Toyobo, Osaka, Japan) using oligo (dT) primers and the resulting cDNA was used as probe for membrane hybridization. Membrane was soaked in 2 × SSC at room temperature for 20 min with shaking prior to hybridization, then 10 ml pre-heated (42°C) ECLTM gold hybridization buffer (GE Healthcare) was added in a plastic bag and kept for 1 h with shaking at 42°C. For probe

labeling, 5 μl cDNA (5 ng RNA) was boiled for 5 min, followed by immediate cooling on ice for 5 min. A same amount (5 μl) of labeling agent and glutaraldehyde were added to the probe and incubated for 15 min at 37°C according to ECL enhanced chemiluminescence DNA labeling and detection kit (GE Healthcare). The labeled probe was added in the hybrid bag and incubated at 42°C overnight with gentle shaking. Probe preparation was repeated twice from independently isolated total RNA, and hybridization was carried out with independently prepared membranes. Signal detection and data analysis After overnight hybridization at 42°C, membrane was washed twice at 42°C with preheated primary wash buffer (0.1 × SSC) followed by two times washing in 2 × SSC at room temperature. Detection buffer was added to the membrane, which was exposed to the x-ray film (Fuji) for 2 h. Scanning was performed with GS-710 Imaging Densitometer (BioRad) and spot intensities were quantified by Image J (available at http//rsb.info.nih.gov/ij). For each gene, signal intensities of two spots were averaged and the mean values were normalized with respect to the actin gene signal intensity of the same membrane. Statistics was performed by t-test to evaluate the significance of changes in the gene expression level. The genes were grouped according to their expression pattern using hierarchical clustering algorithm developed by Eisen et al. (1998), which is based on the average-linkage method of Sokal and Michener (1958) for clustering correlation matrixes such as those used here. Software implements of this algorithm can be obtained from the authors at http://rana.stanford.edu/clustering. RESULTS AND DISCUSSION Respiratory burst in wheat embryos after imbibition Since we decided to conduct our experiment under the total darkness in order to avoid effects of photosynthesis, we first studied the time course of growth and respiratory development in wheat embryos and young seedlings under this condition, which in fact reflects a natural one because seeds are usually sown and germinate beneath the soil. Wheat embryos after germination increased both their shoot and root weight and length continuously during the tested period (Fig. 1). The growth was likely triggered by the start of respiration via mitochondria because energy for biosynthetic processes could exclusively be provided by mitochondrial respiration using stored carbohydrates in this period. To check this, we measured respiration based on the rate of oxygen consumption and estimated the capacity of both cytochrome and alternative pathways. Respiration quickly started within 5 min of hydration of dry embryos and its rate increased markedly during the first day to reach 0.32

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Fig. 1. Shoot and root weight (A) and length (B) of a wheat cultivar CS during the first three days of germination in the dark. All data represent means with standard deviations (vertical lines) (n = 15). Letters (a, b and c) represent significant differences at the 1% level according to Duncan’s test.

nmol O2/min/mg fresh weight of shoot (Fig. 2). Both the cytochrome and alternative pathways contributed to this initial respiratory burst. Later, however, the rate of respiration decreased continuously towards the third day in both pathways. This was in contrast to high plateau lev-

Fig. 2. Development of the respiratory activity during the first three days of wheat germination in the dark. All data represent means with standard deviations (vertical lines) (n = 15) Cyt. path.: cytochrome pathway. Alt. path.: alternative pathway.

els of respiration maintained in 16 h light and 8 h dark conditions (data not shown). The difference can be ascribed to the presence/absence of light illumination and thus to photosynthesis. It is suggested that the initial burst of mitochondrial respiration supports wheat germination and post-germination growth in the dark even after respiration declines and no energy and metabolites are provided by photosynthesis. Development of macroarray system To study changes in the mitochondrial transcriptome profile during the initial 3 days of wheat germination, we devised a mitochondrial macroarray. Because of high homology between the rice and wheat mitochondrial genomes (Notsu et al., 2002; Ogihara et al., 2005) and for achieving high versatility of the macroarray system, we selected nearly all known protein encoding genes and orfs present in the rice mitochondrial genome and four additional protein coding genes unique in wheat. Wheat homolog of rice orf159b is a full size sequence but it is likely a pseudogene because of the presence of stop codons in all 3 reading frames and thus it may or may not be transcribed in wheat. A BLAST search indicated that all other rice orfs were either not present in wheat or present as partial sequences and they were not included in our

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Fig. 3. Reproducibility of the macroarray data. Two different arrays containing the complete set of mitochondrial genes were hybridized with two different cDNAs prepared using 2-day-old samples, and the results were plotted. E1 and E2 represent experiment 1 and 2, respectively. Values represent signal intensity (raw data). Circled point indicates actin transcript.

study. At first, macroarray conditions were optimized. After examining different amounts of target amplicons (genomic) dot-blotted on membranes, 50 ng was selected as an optimum amount. Different amounts of cDNA derived from total RNA were also tested to select 5 ng as satisfactory for all genes. After optimization, we performed hybridization experiments twice using cDNA fragments derived from independently prepared samples. Figure 3 shows an example of linear regression between array hybridization results of the first and second experiment (r = 0.96) using cDNA obtained from 2-day-old samples. Correlation coefficients between all combinations ranged from 0.93 to 0.96, which are nearly equal to ones previously reported for both macroarray and microarray systems (Giege et al., 2005, Wang et al., 2005). The macroarray spots can bind cDNA fragments derived from both the primary and post-transcriptionally modified transcripts. It is thus considered that the devised system is reliable and allows fairly precise quantification of overall transcript abundance. Mitochondrial transcript profiling by macroarray Because the onset of respiration of embryos after imbibition was quite rapid (Fig. 2) and also it has been reported that dry quiescent embryos contain all mitochondrial components required for the initial respiration in sunflower (Attuci et al.,1991), we first tried to quantify the amount of mitochondrial transcripts in dry embryos. Dry embryos of wheat contained transcripts of all genes studied (Fig. 4A), which most likely represented ones synthesized during embryogenesis and stored in dry embryos. A maximum of 6-fold difference was observed

in the amount of individual transcripts in dry embryos. We next determined steady state transcript levels in germinating embryos and growing seedlings. During this stage of germination-through-seedling development, a majority of the mitochondrial genes displayed dynamic changes in their transcript abundance. Much larger variability was observed in the amount of individual transcripts during transition from the first to the second day (sample shown in Fig. 4B), indicating that mitochondrial biogenesis was taking place with de novo synthesis and/or destruction of mitochondrial transcripts. As compared to one-day embryos, transcripts of 13 genes increased in their abundance, those of 3 genes decreased and 13 genes remained constant in 2-day-old germinating embryos. Transcript profiles during the tested period are organized according to their relative abundance using hierarchical clustering. This clustering is based on foldchanges in gene expression at given time points as compared to the previous time points. Four clusters were detected among 29 mitochondrial genes and 5 nuclearencoded mitochondria-targeted genes (Fig. 5). In Fig. 6, changes in transcript profiles of two representative genes in each cluster are shown. The smallest cluster A contained transcripts of two genes (orf176 and nad5) that showed fluctuations with an apparent drop in 2-day-old samples. Sequences similar to rice orf176 are present in mitochondrial genomes of four other cereals, i.e. Sorghum bicolor, Tripsacum dactyloides, Zea mays and wheat (Triticum aestivum). The observed expression in wheat and the ubiquitous presence likely indicates that orf176 is a functional gene. A major cluster B contained 13 genes, among which 9 belonged to the mitochondrial genes encoding subunits of respiratory complexes I, III,

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Fig. 4. Histograms representing the abundance of mitochondrial transcripts in dry embryos (A) and germinating and growing seedlings at 1 day (white bars) and 2 day (black bars) after imbibition (B). Values represent relative signal intensity (raw data). Black circles indicate genes whose steady state transcript levels increased significantly (t-test) from 1 day to 2 day, while white diamonds indicate the opposite change.

IV and V. Two genes in complex IV (cox1 and cox3), one in complex V (atp8) and one involved in cytochrome C biogenesis (ccmFn) belonged to a small subcluster. The amount of all transcripts in cluster B showed a continuous increase to peak at the second day followed by a decrease toward the third day. A sequential increase was observed in respiration, abundance in transcripts of this group and seedling growth. Thus the respiratory burst took place first (Fig. 2), followed by the increase in transcript abundance (Fig. 6B) and then by the increase in seedling growth (Fig. 1). This suggests that the rapid respiratory burst is supported by the stored respiratory components, which was followed by de novo synthesis of mRNAs leading to subsequent seedling growth. The increase in the transcript abundance before the start of seedling growth indicates that substrate availability might be one regulatory factor of mitochondrial gene expression. Similar general increase of some mitochondrially encoded transcripts after 48h of germination was

reported in maize (Logan et al., 2001). Because there was no switch to photosynthesis under the experimental condition, the decrease in respiration might be a mechanism of compensation for maintaining seedling growth when the carbohydrate reserves decline. Cluster C contained one mitochondrial (rps7) and two nuclear genes (TamRPL5 and TamRPL11) encoding mitochondrial ribosomal protein subunits. These transcripts showed a continuous increase during the tested period. As a whole, nearly two thirds of the mitochondria-encoded transcripts belonged to A, B and C clusters, while the second largest cluster D contained the remaining one third. Genes in cluster D were unique in that the amount of individual transcripts remained at constant and high levels throughout the tested period. Five genes encoding subunits of mitochondrial respiratory complex I (nad4L, nad6 and nad9) and V (atp4 and atp6), a gene (rps12) encoding a ribosomal protein subunit and Whlp, which is a wheat homolog of Arabidopsis HLP encoding a mitochondrial

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Fig. 5. Hierarchical clustering of 29 mitochondria-encoded and 5 nuclear encoded (boldface letters) and mitochondria-targeted genes, showing changes in the expression pattern during wheat embryo germination. Actin gene was used as a between array control. Green represents decreases (–1 to –2.5) and red increases (+1 to +2.5) in the steady state RNA abundance as compared to that of the previous day during the tested period. Letters A to D indicate 4 major expression patterns identified by the clustering program (see Results and Discussion). Each gene is represented by a single row of colored boxes, and each condition is represented by a single column.

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Fig. 6. Four major clusters of mitochondrial transcripts and a nuclear encoded and mitochondria-targeted transcript showing distinguishable expression profiles during wheat embryo germination. In each cluster (A to D), representative genes are shown. TamRPL5 is a nuclear encoded and mitochondria-targeted transcript.

ribosomal protein subunit L14 (Mizumoto et al., 2004), were included in cluster D. This cluster also contained transcripts of a nuclear gene Ubiquitin and a nuclear encoded mitochondrial manganese SOD gene (Mn-SOD). It was further noted that all transcripts of two mitochondrial and two nuclear genes encoding mitochondrial ribosomal protein subunits belonged to either cluster C or D, indicative of active mitochondrial protein synthesis as suggested during germination and early seedling development in wheat (Li-Pook-Than et al., 2004). Howell et al. (2006) reported that in germinating rice embryos the amount of transcripts of mitochondrial genes encoding subunits of the respiratory chain component

F1α (complex V) and cox2 (complex IV) peaked at 48 h post-imbibition, while transcripts of the nad9 (Complex I) and cob (Complex III) peaked earlier at 8 h postimbibition and then slightly decreased in their amount. Although transcript profiles of individual genes in rice and wheat are different, partly because of differences in the experimental conditions, a sequential order of transcript accumulation needed for the synthesis of mitochondrial components (Fig. 6) appears to be a general phenomenon. Transcript of atp6 was stably expressed in germinating wheat embryos (Fig. 6), agreeing with the previous result in maize (Logan et al., 2001) and wheat (Li-Pook-Than et al., 2004).

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It was reported that a region of wheat mitochondrial genome consisting of nad3-rps12-orf229-atp8 (formerly orf156) is transcribed into a single polycistronic messenger RNA (Gualberto et al., 1991; Kitagawa et al., 2003). As we studied three genes from this transcription unit (nad3, rps12 and atp8), we expected that all showed the same trend in their transcript accumulation. We found that nad3 and rps12 transcripts belonged to the same subcluster in cluster D. However, though a reason remains unknown, atp8 transcript was found in cluster B. Interesting result was obtained for another polycistronic unit in wheat nad6-nad1 exon 4 (Haouazine et al., 1993). Wheat nad1 gene consists of five exons, which are scattered throughout wheat mitochondrial genome, and exons 4 and 5 are separated by trans-spliced intron (Chapdelaine and Bonen, 1991). Our result indicated that nad6 showed constant steady state transcript abundance but the level of nad1 transcript decreased during the third day. This result might be due to the use of exon 3 as probe for nad1 gene. nad1 exon 3 probe likely detected transcripts derived from unit containing the exon 3 region before the trans-splicing event as well as mature transcripts, resulting in the decrease in the level of nad1 exon 3 during the third day. Although a reason for this difference in transcript levels between different transcriptional units carrying exons for one mature transcript is unknown, it is suggested that wheat mitochondria transcriptional regulation is complex and involves many steps. Therefore, different levels of transcript accumulation can be obtained by macroarray, RT-PCR and northern blot according to selected subunits of a given gene used for measuring. Wheat mitochondrial nad 5 gene also has been reported to be dispersed and its 5 exons locate 3 widely separated regions of the genome that are independently transcribed (Pereira de Souza et al., 1991). nad2 is another known trans-split gene with five exons locating in two distant regions (MorawalaPatell et al., 1992). rps3-rpl16 transcription unit also gives rise to polycistronic mRNA in wheat (Li-Pook-Than et al., 2004), but we did not include this transcription unit in our array. Respiration via the alternative pathway followed the same trend as the overall respiration in wheat during the post-imbibition period in the dark (Fig. 2). We applied the macroarray system using Waox1a as a probe to study wheat aox gene expression, but could not detect corresponding transcripts throughtout the test period. Quantitative real-time PCR analysis, however, showed a constant steady-state level of this transcript, although under different growth conditions (data not shown), thus suggested its low abundance. For some unknown reason, mitochondrial cox2 gene was also not detected by our macroarray, although a blast analysis and alignment of the rice and wheat cox2 gene showed a considerable homology (98.9%), which is high enough to allow for sta-

ble hybridization. Real-time PCR analysis again showed that cox2 was transcribed throughout the tested period (data not shown). Our knowledge about the regulation of mitochondrial transcriptome is just at the infant stage. Not only messenger RNA transcription but also RNA-processing events such as C-to-U type RNA editing, cis/trans-splicing and maturation of transcript termini are all involved in this regulation that is coupled with mitochondrial biogenesis (Li-Pook-Than et al., 2004). The macroarray system we developed can provide useful means to get first insights into the regulation and role of mitochondrial transcriptome associated with mitochondrial biogenesis during the various developmental stages under both normal and stressed conditions in cereals. However, as indicated in Fig. 4 by the detected amount of rice orf25 and wheat atp4 transcripts (both are now known as the same gene, see Table 1) and of wheat nad3, rps12 and atp8 transcripts (these are polycistronic; Gualberto et al., 1991), our present macroarray system gave some inconsistency and uncertainty in quantification. To improve reliability of the macroarray system, further technical refinement is required, particularly in the probe-spotting procedure. We thank Dr. S. Takumi for his advices and help in the macroarray experiment and Dr. T. Nishikawa for providing primer information of rice mitochondrial genome. S. K. is a recipient of Japanese Ministry of Education predoctoral scholarship and N. G. N. is a recipient of Japanese Society for the Promotion of Science postdoctoral fellowship; grant no. P05187. Contribution no. 184 from the laboratory of Plant Genetics, Graduate School of Agricultural Science, Kobe University.

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